In the absence of activation of certain inflammasomes, caspase-1 is primarily detected as pro-caspase-1 (p45) in cell lysates. However, upon activation of certain inflammasomes (such as NLRP3), the pro-caspase-1 is proteolytically cleaved to detectable p20 and p10 forms. These forms are detectable in cell lysates at earlier time (after activation of an inflammasome). However, at a later time, the bulk of these forms are secreted and are detected in cell culture medium or supernatants upon precipitation of proteins. Antibodies to detect murine pro-caspase-1 and its cleaved forms are available from limited sources. Antibodies to caspase-1 from Invitrogen has worked well for us in immunoblotting (see our several publications). Unfortunately, the supplier has discontinued to market antibodies to caspase-1 that worked well. Therefore, it may be challenging to find usable antibodies from other commercial suppliers.

Try one of the fluorogenic substrates available on the market. This is the simplest issue when looking for activity.

If you still want to detect protein in the sup itself (and not the activity as comrades have suggested here :) ) then try precipitation (method of Wessel fluege, but not TCA!!!) of it or run through Amicon Ultra to concentrate and then use for WB.

Treat cells with LPS 2ug/ml for 6hrs and 5mM ATP for 2hrs in medium with heat inactivated FCS. Abcam anti caspase 1 antibody detects p20, R&D antibody detects uncleaved 45KD band. It worked in kidney cells. Detecting in supernatants by TCA precipitation does not exactly works. Good Luck!!!

If you have the cytometer you can us e.g. FAM FLICA™ Caspase 1 Assay Kit

Appears to be very interesting and not complicated method :)

The suggestions above are good ones. I feel that historically, variation in the ability of researchers to observe casp-1 activation by LPS has been due to 2 not-necessarily mutually-exclusive factors. 1) the amount of LPS used. I've seen papers using 10-100µg/ml in some cases. Too much IMO. 2) and the source/quality of the LPS used. Many older papers, and even some new ones still use LPS from Sigma which has been observed to contain inflammasome agonists. This highly cited paper (below), but IMO under-read paper sheds light on the topic of LPS contaminants and inflammasome activation. If your using ultra-pure LPS then your already on the right track to produce quality data for the field.

Identification of bacterial muramyl dipeptide as activator of the NALP3/cryopyrin inflammasome

F Martinon, L Agostini, E Meylan, J Tschopp - Current Biology, 2004

Western blotting, I have done that

The antibody from Invitrogen detects the murine caspase-1 really well in immunoblotting (see our papers). Good luck with your detection.

I suggest Immunophuorescence on the culture cell, or you can also try to use crio-immuneelectronmicroscopy and make the immunolabeling with gold particules conjugated with you antibody.

When I was trying to detect caspases (3, 8, 9) with WB, I couldn't understand why I didn't get any cleaved bands. Then I realised I had to wait a specific time point after my treatment to see the band appear. I suggest you to do a time point experiment and get lysates at different times. Mine appeared after 16hrs, but it may change with your treatment or with your cell type. But then western worked very well at that time point. Good luck.

Thanks all. I've been looking at cell lysates at 0, 1hr, 6hr and o/n stimulation with 25 ng/ml LPS (sigma L6529) and can see the procaspase-1 form with no problem. I've run a gel on the supernatants as well now, and think I may be seeing the p20.

I've seen the p20 reported as a band anywhere from 15-25 kDa. Any consensus here on which is correct?

Initiator caspases, like caspase-1, do not require cleavage for activation, thus cleavage does not really indicate that the caspase is activated. At present the best indication of initiator caspase activation is obtained using a caspase affinity ligand, bVAD, which binds to activated caspases irreversibly. The caspase-bVAD complex can then be pulled out with streptavidin, and western blot used to detect the caspase pulled out - for initiators you will detect full length. Key to the method is to treat the cells with bVAD before applying LPS so that the bVAD is available to bind the caspase as it is activated. See Tu et al. Nature Cell Biology, 8:72-77, 2006 for the method. We have used this successfully in primary neurons and in vivo in the CNS.

I think that The best way for detection of caspase-1 is the western blot.

hi Jason, have you successfully detected caspase-1 p20 yet? I have the same problem now.I can always detect pro-caspase-1(45kda) but can't find any band near 20kda. Just wondering how you did wb in supernatant ?

and Jason ,also look at this:

https://www.researchgate.net/post/I_cant_detect_caspase-1_activation_band_p10_in_my_WB

I too had problems until started using the antibody from Adipogen (AG-20B-0042). Load max supernatents

In the absence of activation of certain inflammasomes, caspase-1 is primarily detected as pro-caspase-1 (p45) in cell lysates. However, upon activation of certain inflammasomes (such as NLRP3), the pro-caspase-1 is proteolytically cleaved to detectable p20 and p10 forms. These forms are detectable in cell lysates at earlier time (after activation of an inflammasome). However, at a later time, the bulk of these forms are secreted and are detected in cell culture medium or supernatants upon precipitation of proteins. Antibodies to detect murine pro-caspase-1 and its cleaved forms are available from limited sources. Antibodies to caspase-1 from Invitrogen has worked well for us in immunoblotting (see our several publications). Unfortunately, the supplier has discontinued to market antibodies to caspase-1 that worked well. Therefore, it may be challenging to find usable antibodies from other commercial suppliers.

Timing, indeed, is one of the most critical components here.